1. Field of the Invention
This invention relates to antimicrobial agents and their use.
2. Discussion of the Related Art
Antimicrobial agents are used in the treatment of infections, particularly of the gastrointestinal tract. Gastrointestinal infections affect millions of people world-wide, especially children. Infantile gastroenteritis affects more than 20 million children below twelve months age worldwide annually and considered a leading cause of mortality. Intestinal infections in children, unassociated with antibiotic use or hospital stays, can cause chronic diarrhea and failure to grow. (E.g., T. E. Liston, Clostridium difficile toxin associated with chronic diarrhea and failure to gain weight, Clin. Pediatr. (Phila.) 22(6):458-60 [1983]). Toxic shock, or bacteraemia and subsequent sepsis are other possible complications of intestinal infection. (See, e.g., P. Naaber et al., Bacterial translocation, intestinal microflora and morphological changes of intestinal mucosa in experimental models of Clostridium difficile infection, J. Med. Microbiol. 47(7):591-98 [1998]). Enterotoxigenic bacterial strains are linked with a significant number of cases of antibiotic-associated diarrhea, especially among the elderly, children, and infants. Gastrointestinal infections also pose an increasing health hazard in hospital settings.
Of course, bacteria inhabit healthy intestines to the benefit of their human and animal hosts. But pathogenic bacterial strains and opportunistic pathogens that can infect immunocompromised hosts have an adverse disease-causing effect.
Prominent agents of microbial disease include Clostridium species, especially C. difficile and C. perfringens. Clostridium species are gram-positive, spore-forming anaerobes; some strains that colonize the human intestines can, under certain circumstances, release potent protein exotoxins that induce inflammation of the intestinal mucosa. (M. L. Job and N. F. Jacobs, Jr., Drug-induced Clostridium difficile-associated disease, Drug Saf.17(1):3746 [1997]). For example, antibiotics and other chemotherapeutic agents can induce the expression of Toxins A and B from Clostridium difficile. (B. A. Cunha [1998]). Agents known to have a high potential to induce C. difficile-associated disease are aminopenicillins, cephalosporins and clindamycin. (M. L. Job and N. F. Jacobs, Jr., Drug-induced Clostridium difficile-associated disease, Drug. Saf. 17(1):37-46 [1997]; Y. Hutin et al., Prevalence of and risk factors for Clostridium difficile colonization at admission to an infectious diseases ward, Clin. Infect. Dis. 24(5):920-24 [1997]; C. D. Settle and M. H. Wilcox [1996]).
In developed countries, the great majority of cases of C. difficile infection are hospital-acquired, and the number of nosocomial clostridial infections is reported to be rising. (C. D. Settle and M. H. Wilcox, Review article: antibiotic-induced Clostridium difficile infection, Aliment. Pharmacol. Ther. 10(6): 83541[1996]; J. S. Brazier, The epidemiology and typing of Clostridium difficile, J. Antimicrob. Chemother. 41 Suppl. C: 47-57 [1998]; S. Tabaqchali and M. Wilks, Epidemiological aspects of infections caused by Bacteroides fragilis and Clostridium difficile, Eur. J. Clin. Microbiol. Infect. Dis. 11(11): 1049-57 [1992]; C. R. Clabots et al., Acquisition of Clostridium difficile by hospitalized patients: evidence for colonized new admissions as a source of infection, J. Infect. Dis. 166(3):561-67 [1992]).
A nosocomial pleural infection with C. difficile, following surgical insertion of a chest drain has also been reported (A. J. Simpson et al., Nosocomial empyema caused by Clostridium difficile, J. Clin. Pathol. 49(2):172-73 [1996]), but intestinal infections are the greatest problem.
Nosocomial diarrhea due to gastrointestinal infection with C. difficile has become a major health care problem, causing 20-30% of all nosocomial diarrheas and affecting up to 8% of hospitalized patients. (L. R. Peterson and P. J. Kelly, The role of the clinical microbiology laboratory in the management of Clostridia difficile-associated diarrhea, Infect. Dis. Clin. North Am. 7(2):277-93 [1993]). Clostridium difficile is considered to be the premier cause of diarrhea among hospitalized patients. (M. Delmee et al., Treatment of Clostridium difficile colitis. Summary of a round table held in Brussels on Mar. 3, 1994, Acta Clin. Belg. 50(2):114-116 [1995]).
In developing countries, C. difficile is also thought to be a causal agent of wide-spread acute diarrheal disease. (S. K. Niyogi et al., Prevalence of Clostridium difficile in hospitalized patients with acute diarrhea in Calcutta, J. Diarrhoeal Dis. Res. 9(1):16-19 [1991]; S. Q. Akhtar, Isolation of Clostridium difficile from diarrhea patients in Bangladesh, J. Trop. Med. Hyg. 90(4):189-92 [1987]).
Enterotoxigenic strains of C. perfringens are linked with a significant number of cases of antibiotic-associated diarrhea, especially among elderly hospitalized patients, children, and infants. (A. Wada et al., Nosocomial diarrhea in the elderly due to enterotoxigenic Clostridium perfringens, Microbiol. Immunol. 40(10):767-71 [1996]; M. M. Brett et al., Detection of Clostridium perfringens and its enterotoxin in cases of sporadic diarrhea, J. Clin. Pathol. 45(7):609-11 [1992]; S. C. Samuel et al., An investigation into Clostridium perfringens enterotoxin-associated diarrhea, J. Hosp. Infect. 18(3):219-30 [1991]; S. P. Boriello et al., Epidemiology of diarrhea caused by enterotoxigenic Clostridium perfringens, J. Med. Microbiol. 20(3):363-72 [1985]; R. Willliams et al., Diarrhoea due to entertoxigenic Clostridium perfringens: clinical features and management of a cluster of 10 cases, Age Ageing 14(5):296-302 [1985]). Clostridium perfringens has been implicated as a possible contributor to sudden infant death syndrome (SIDS) in susceptible infants. (R. R. Meer et al., Human disease associated with Clostridium perfringens enterotoxin, Rev. Environ. Contam. Toxicol. 150:75-94 [1997]).
Clostridium perfringens is well known as a causative agent of non-gastrointestinal gangrene, a special problem for many elderly and diabetic patients with poor blood circulation. But also in more extreme cases of gastrointestinal infection, C. perfringens can cause enteritis necroticans, a gangrene of the bowel resulting in necrosis, sepsis, and hemolysis, in humans and domesticated animals. (L. E. Clarke et al., Enteritis necroticans with midgut necrosis caused by Clostridium perfringens, Arch. Surg. 129(5):557-60 [1994]; D. Bueschel et al., Enterotoxigenic Clostridium perfringens type A necrotic enteritis in a foal, J. Am. Vet. Med. Assoc. 213(9):1305-07 [1998]; E. G. Pearson et al., Hemorrhagic enteritis caused by Clostridium perfringens type C in a foal, J. Am. Vet. Med. 188(11): 1309-10 [1986]; F. Al-Sheikhy and R. B. Truscott, The interaction of clostridium perfringens and its toxins in the production of necrotic enteritis of chickens, Avian Dis. 21(2):256-63 [1977]).
Although rare in developed countries, clostridial enteritis necroticans in humans is more common in some developing countries. (D. A. Watson et al., Pig-bel but no pig: enteritis necroticans acquired in Australia, Med. J. Aust. 155(1):47-50 [1991]). In New Guinea, enteritis necroticans, known locally as pigbel, has been a major cause of illness and death among children. (G. W. Lawrence et al., Impact of active immunisation against enteritis necroticans in Papua New Guinea, Lancet 336(8724):1165-67 [1990]). Clostridium perfringens type C, the etiologic agent of enteritis necroticans, was also isolated from Bangladeshis with bloody or watery diarrheal illness. (F. P. van Loon et al., Clostridium perfringens type C in bloody and watery diarrhea in Bangladesh, Trop. Geogr. Med. 42(2):123-27 [1990]).
Entertoxigenic strains of C. perfringens have also been linked to nosocomial and non-nosocomial outbreaks of food poisoning, due to heat-resistant spores and a rapid growth rate in warm food. (A. M. Pollack and P. M. Whitty, Outbreak of Clostridium perfringens food poisoning, J. Hosp. Infect. 17(3):179-86 [1991]; M. Van Darnme-Jongsten et al., Synthetic DNA probes for detection of enterotoxigenic Clostridium perfringens strains isolated from outbreaks of food poisoning, J. Clin. Microbiol. 28(1):131-33 [1990]).
Spores of Clostridium botulinum germinating in warm food can cause another form of food poisoning called botulism. Growing particularly in non-acidic foods lacking nitrites, and protected from oxygen, the vegetative cells of C. botulinum release an exotoxin that when consumed with the food is activated by trypsin in the stomach, and is absorbed intact by the blood stream. The exotoxin binds to nerve cells, preventing the release of the neurotransmitter acetylcholine. Resulting symptoms of botulism include blurred vision, difficulty in swallowing and speaking, and increasing muscular weakness, and usually nausea and vomiting. Death often results from paralysis of the muscles required for breathing. (R. Y. Stanier et al., The Microbial World, 5th ed., Prentice Hall, Englewood Cliffs, N.J., pp. 626-27 [1986]). Clostridium botulinum sometimes colonizes the intestines of infants and can cause infantile botulism, which can lead to respiratory paralysis and sudden infant death.
Botulism is a problem for the food packaging industry. Spores of C. botulinum may not be killed if canning is done at too low a temperature. High temperature autoclave treatment may be unsuitable for some foods Mayonnaise and other non-acidic foods are particularly prone to foster the growth of C. botulinum. Now with increasing health concerns about the use of nitrite as a food preservative, alternative antimicrobial agents are needed against the growth of C. botulinum and other food poisoning bacterial pathogens.
Of course, a wide variety of microbes, beside pathogenic Clostridium spp., poses problems of contamination and infection. These include bacteria such as enterotoxigenic Escherichia coli, enteropathogenic Escherichia coli, verotoxic Escherichia coli, including serotype O157:H7, Shigella dysenteriae, Shigella flexneri, Salmonella typhimurium, Salmonella abony, Salmonella dublin, Salmonella hartford, Salmonella kentucky, Salmonella panama, Salmonella pullorum, Salmonella rostock, Salmonella thompson, Salmonella virschow, Campylobacter jejuni, Aeromonas hydrophila, Staphylococcus aureus, Staphylococcus hyicus, Staphylococcus epidermidis, Staphylococcus hominis, Staphylococcus warneri, Staphylococcus xylosus, Staphylococcus chromogenes, Bacillus cereus, Bacillus subtilis, Candida albicans, and such radiation-resistant bacteria as: Brochothrix thermospacta, Bacillus pumilus, Enterococcus faecium, Deinococcus radiopugnans, Deinococcus radiodurans, Deinobacter grandis, Acinetobacter radioresistens, Methylobacterium radiotolerans, as well as other kinds of bacteria. Various pathogenic fungi, protozoa and viruses are also microbes that are often difficult to treat with known antimicrobial agents.
Antimicrobial agents with selective toxicity for a specific spectrum or range of pathogenic microorganisms are well known in the art. One class of antimicrobial agents is the antibiotics, which are compounds, synthesized and excreted by various microorganisms, that are selectively toxic to other microorganisms, specifically bacteria. In addition, some antibiotics can be artificially modified to produce antimicrobial agents that are more effective and/or more able to overcome antibiotic resistance.
Antimicrobial agents with selective toxicity for a specific spectrum or range of pathogenic microorganisms are well known in the art. One class of antimicrobial agents is the antibiotics, which are compounds, synthesized and excreted by various microorganisms, that are selectively toxic to other microorganisms, specifically bacteria. In addition, some antibiotics can be artificially modified to produce antimicrobial agents that are more effective and/or more able to overcome antibiotic resistance.
One of the most commonly used antibiotics for the treatment of gastrointestinal infections or bacterial overgrowths is vancomycin. Like many antimicrobial agents, vancomycin is prohibitively expensive, especially for developing countries, and there are concerns about the rapid development of vancomycin-resistance among pathogenic Clostridium, Enterococcus, Pediococcus, Citrobacter, Klebsiella, Enterobacter, and Staphylococcus species, because the plasmid-borne vancomycin resistance gene (VanR) is readily transmissible. (ASHP therapeutic position statement on the preferential use of metronidazole for the treatment of Clostridium difficile-associated disease, Am. J. Health Syst. Pharm. 55(13): 1407-1 [1998]; S. H. Cohen et al., Isolation of a toxin B-deficient mutant strain of Clostridium difficile in a case of recurrent C. difficile-associated diarrhea, Clin. Infect. Dis. 26(2): 1250 [1998]; C. Edlund et al., Effect of vancomycin on intestinal flora of patients who previously received antimicrobial therapy, Clin. Infect. Dis. 25(3):729-32 [1997]; C. A. O'Donovan et al., Enteric eradication of vancomycin-resistant Enterococcus faecium with oral bacitracin, Diagn. Microbiol. Infect. Dis. 18(2):105-09 [1994]; E. Yamaguchi et al., Colonization pattern of vancomycin-resistant Enterococcus faecium, Am. J. Infect. Control 22(4):202-06 [1994]; C. P. Kelly and J. T. LaMont [1998]). The phenomenon of antibiotic resistance, by no means limited to vancomycin-resistance, is an increasing public health problem.
Accordingly, there remains a definite need for a modestly priced antimicrobial agent for treating gastrointestinal and other infections, without the commonly unpleasant side effects and bacterial resistance often associated with vancomycin and other antibiotics.
Meat animals and egg laying hens often carry, among their native gastrointestinal microflora and/or within their lymph nodes, bacteria associated with food-poisoning and gastrointestinal disease in humans, for example various Salmonella species. (E.g., Letellier, A. et al., Assessment of various treatments to reduce carriage of Salmonella in swine, Can. J. Vet. Res. 64(1):27-31 [2000]). Some pathogenic bacteria, such as Salmonella enteritidis, can invade the reproductive organs of egg layers and ultimately contaminate egg contents. (E.g., Seo, K. H. et al., Combined effect of antibiotic and competitive exclusion treatment on Salmonella enteritidis fecal shedding in molted laying hens, J. Food Prot. 63(4):545-48 [2000]).
With respect to the meat, the edible tissues of a healthy meat animal are essentially sterile prior to slaughter. Various innate host defense mechanisms at the external and internal organ surfaces create an effective barrier and prevent microorganisms from invading the tissues of a live animal. As soon as the animal is slaughtered, however, the natural defenses against invading microbes virtually disappear, and the exposed tissues become highly susceptible to microbial colonization and proliferation. Meat of the freshly slaughtered animal is prone to contamination with a variety of bacterial species, influenced by the degree of sanitation practiced during the meat processing and packing operations.
The economic impact of food-borne pathogenic outbreaks and the shorter than desired shelf life of refrigerated products, even vacuum packaged refrigerated products, has necessitated the search for an effective antimicrobial system for the meat industry. The recent occurrence of verotoxic Escherichia coli (E. coli) serotype O157:H7 in ground beef causing hemolytic uremic syndrome highlighted this longstanding problem, in foods and, especially, meats. It has prompted a major review of safety issues in the food industry and a call for improved methods for preventing microbial contamination. Various methods are currently in practice to control E. coli and other microbial contamination in foods, but, unfortunately, they suffer from a variety of drawbacks.
For example, in the meat industry, acid washing of beef carcasses is currently being employed as a microbial intervention. However, recent studies have shown that certain types of E. coli, such as the verotoxic strains of serotype O157:H7 and vancomycin-resistant strains of Enterococcus faecium, can survive acid conditions, while at the same time produce harmful toxins. The meat industry is also irradiating meat in an attempt to control pathogens and food spoilage organisms. However, studies have shown that although irradiation appears to be effective at killing some types of Escherichia coli, there are still various other microorganisms, including strains of Brochothrix thermospacta and Bacillus pumilus, known to be radiation resistant and thus are able to survive such processes. Irradiation also can produce undesirable changes in the texture and/or organoleptic quality of beef. Further, both of these methods are cidal processes that kill microorganisms leaving endotoxins, microbial debris and other proinflammatory substances which can cause undesirable immunological reactions in the host. Finally, neither of these methods excludes the possibility of post-processing contamination once the beef is treated for microbial contaminants.
In addition to food-borne pathogens, microbial spoilage of packaged foods, including fresh meats and vegetable foods, is a significant concern to the food industry. Under certain conditions, it is possible to control microbes, including enteric pathogens, using such well known antimicrobial agents as acids, salts, oxidative agents, antibiotics, bacteriocins, and the like. Typically, the mode of action of these agents is “cidal”—the direct killing of the microbes, or “stasis”—the inhibition of microbial growth/multiplication. Another mode of action for conventional antimicrobial agents is opsonization. The agents intervene by promoting microbial phagocytosis by macrophages.
Certain cellular research relating to the mechanisms of microbial biosurface interactions has led to the identification of another mode of action, microbial blocking, and a new class of antimicrobial agents, microbial blocking agents (MBAs). MBAs are naturally occurring biological substances that block microbial adhesion-colonization, retard growth-multiplication, and neutralize the adverse effects of proinflammatory cell debris.
It has not proved possible to apply such microbial blocking agents during meat packing or other food processing conditions, because of the difficulty of delivering a biofunctionally active and structurally stable MBA to the food product to be treated. The difficulty is compounded when the food product is a meat product, because a controlled milieu is required for a broad-spectrum activity of MBA to block various microorganisms on a chemically complex and heterogenous meat tissue.
Breast-fed infants are better protected against various gastrointestinal infections than the formula-fed infants. Lactoferrin is one of the major antimicrobial systems in milk and colostrum and has been credited for protection of newborns against gastrointestinal illness. However, deficiency or dysfunction of lactoferrin and/or other antimicrobial systems may exist in certain breast milks depending on the nutritional and health status of feeding mothers.
Lactoferrin (LF) is an iron-binding glycoprotein present in milk and various mammalian secretions (e.g. saliva, tears, mucus, and seminal fluids). Crystallographic studies of LF indicate a bilobate structure (N-terminus and C-terminus lobes) with one iron-binding site in each lobe. LF has ability to reversibly bind two Fe3+ ions per lobe in coordination with two CO32− ions. LF can release the bound iron in a fully reversible manner, either on exposure to lowered pH (below 4.0) or on receptor binding. This high affinity for iron is linked to many of its biological functions including antimicrobial effects. Various laboratory studies have reported that the structural integrity of LF is critical for its antimicrobial effects against bacteria, fungi, protozoa, and viruses.
However, the activity of LF, like the activity of most proteins, is highly dependent on the three-dimensional or tertiary structure of the protein. If the protein does not have the proper conformation its activity is diminished or lost. LF's instability limits it usefulness. Milieu conditions such as metals (iron in particular), carbonic ions, salts, pH and conductivity affect the antimicrobial properties of LF. In addition, protein isolation procedures, storage, freezing-thawing, can adversely affect the biofunctionality of LF. Consequently, before LF can be used for commercial application, it would be expected to become denatured or inactivated, and lose its antimicrobial properties.
In fact, under certain conditions, when the LF molecule is degraded or denatured, cationic peptide fragments are generated. These cationic peptide fragments exhibit a non-specific antimicrobial activity, making them absolutely unsuitable as an ingredient in a food product. The consumer of a food product does not want to ingest a non-specific antimicrobial agent, because of the agent's adverse affect on the desirable microbes always present in a human body, particularly within the gastrointestinal tract.
Thus, an antimicrobial agent is needed for blocking microbial contamination in foods, meats and seafood in particular, on non-biological surfaces, on biological surfaces, and in biological fluids, whether in vivo or in vitro, that does not pose the undesired affects of cidal antimicrobial systems but that also exhibits carry through properties for the prevention of post-processing contamination.